Clathrin and the coats it forms were discovered nearly 30 years ago. Since then, numerous biochemical and cell biological studies have made the behavior of clathrin coats a paradigm for many aspects of membrane traffic. These approaches, however powerful, can only represent the averaged properties of objects within a heterogeneous population. They cannot provide information about the behavior of a specific coated pit or vesicle. [unreadable] [unreadable] To work out the mechanism underlying formation of clathrin-coated pits, it is now crucial to figure out when, for how long and in what quantity are specific proteins recruited during the life of a coated pit. Recent developments of fluorescent live-cell imaging techniques geared for singlemolecule detection, used in combination with object-identification algorithms, now allow sufficient temporal and spatial resolution to follow the life of a single coated pit. To use this powerful approach, we have developed and implemented over the past two years light-microscopic techniques to analyze the clathrin pathway in living cells. We have shown that it is possible to gather quantitative, "single-object" data from hundreds of uniquely identified clathrin coated pits and coated vesicles, tracked while they are assembling, recruiting cargo and uncoating, using as probes clathrin, AP-2 and dynamin fused to fluorescent EGFP or to mRFP, and fluorescently tagged cargo such as transferrin, LDL and reovirus. [unreadable] [unreadable] In this new project we extend the scope of our previous inquiry to include early steps in the formation and assembly of endocytic clathrin-coated vesicles at the plasma membrane (Aim 1), uncoating on endocytic clathrin structures (Aim 2), and assembling of clathrin coats in endosomes and the trans Golgi network (Aim 3). [unreadable] [unreadable]